ABSTRACT Leukocytes defend the body against infections and tumors. Critical for their job, they are highly motile, using chemotaxis, or migration along chemical gradients, to reach sites of injury and infection. Once there, they can respond to the same chemical signals with cytotoxic and inflammatory responses including degranulation. Leukocyte responses are critical for fighting infections, but over-activity can cause tissue and organ damage and is associated with inflammatory diseases. Additionally, leukocytes are central players in emerging cell-based immunotherapies to treat cancer. These strategies include both efforts to mobilize the body's own leukocytes for tumor destruction and to engineer leukocytes with synthetic receptors specifically targeting tumor cells. While these therapies have shown immense promise, they have also been limited by dangerous side effects from excessive immune responses and insufficient activity towards solid tumors. Maximizing activity against cancer cells, while minimizing nonspecific activity is critical for the effectiveness of these therapies. Synthetic control of chemotaxis could directly address this challenge. However, current efforts to modulate or engineer chemotaxis are limited because many of the basic mechanisms controlling chemotaxis remain unclear. We still lack molecular mechanisms explaining how directionality, response sensitivity, and prioritization among chemoattractants are achieved, and how the signaling pathways controlling chemotaxis and degranulation diverge. A major obstacle to progress has been the complexity of the leukocyte signaling machinery, which involves many highly interconnected components, and controls multiple different cellular responses. Our overall objective is to understand pathway specialization within the chemoattractant signaling network so that we can design synthetic methods for precise and specific control of leukocyte behavior in vivo. In particular, we aim to discriminate the pathways and mechanisms controlling cell directionality, sensitivity of chemotaxis, cross-talk between receptors, and degranulation. To do this, we will break down the complexity of the signaling network using systematic genetic approaches that have been largely absent from the field, including a technique we recently developed for live-cell imaging of chemotaxis in high-throughput, a genetic interaction map, and a panel of genome-wide analyses. These results will serve as a map for the design of synthetic receptors, gene perturbations, and pharmacological treatments to engineer leukocytes with controllable and reversible chemotaxis, and with separable control of migration and degranulation. Finally, we will test our engineering strategies in live zebrafish embryos.